Purification and characterization of alkaline phosphatase from pseudomonas aeruginosa 50071

Alkaline phosphatase was purified to homogeneity from Pseudomonas aeruginosa 50071 starting with heat treatment at 60°C in the presence of Co[2]+ and ammonium sulphate fractionation, followed by separation on DEAE-Sephadex A50 and G-100 Sephadex gel filtration. The enzyme was purified 468 fold and showed a final specific activity of 51.5 unit/mg protein with a yield 42%. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] of the purified enzyme revealed a single protein band of molecular weight 82 KDa. The molecular weight of the native enzyme was calculated to be 158 KDa, as determined by gel filtration, which approximated to two subunits together [164 KDa] suggesting a homogeneous dimeric structure of the enzyme. The enzyme showed a maximum activity at pH 9.5 when incubated at 37°C. A Lineweaver-Burk analysis gave a K[m] of 2.1 mM and V[max] of 8.0 U/ml. The enzyme activity was enhanced by addition of CoCl[2] and ZnCl[2] together. The amino acid composition of the purified enzyme was also determined

Properties of a constitutive alkaline phosphatase from strain 74A of the mold Neurospora crassa

A constitutive alkaline phosphatase was purified to apparent homogeneity as determined by polyacrylamide gel electrophoresis from mycelia of the wild strain 74A of the mold Neurospora crassa, after growth on acetate and in the presence of saturating amounts of inorganic phosphate (Pi) for 72 h at 30ºC. The molecular mass was 58 kDa and 56 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. This monomeric enzyme shows an apparent optimum pH ranging from 9.5 to 10.5 and Michaelis kinetics for the hydrolysis of p-nitrophenyl phosphate (the Km and Hill coefficient values were 0.35 mM and 1.01, respectively), alpha-naphthyl phosphate (the Km and Hill coefficient values were 0.44 mM and 0.97, respectively), ß-glycerol phosphate (the Km and Hill coefficient values were 2.46 mM and 1.01, respectively) and L-histidinol phosphate (the Km and Hill coefficient values were 0.47 mM and 0.94, respectively) at pH 8.9. The purified enzyme is activated by Mg2+, Zn2+ and Tris-HCl buffer, and is inhibited by Be2+, histidine and EDTA. Also, 0.3 M Tris-HCl buffer protected the purified enzyme against heat inactivation at 70ºC(half-life of 19.0 min, k = 0.036 min-1) as compared to 0.3 M CHES (half-life of 2.3 min, k = 0.392 min-1) in the same experiment

Fosfatasa alcalina placentaria de alto peso molecular en plasma de mujeres embarazadas durante el último trimestre de gestación / Placental alkaline phosphatase of high molecular weight in plasma of pregnant women in the last trimestre of gestation

En el plasma humano pueden encontrarse las isoenzimas ósea, hepática e intestinal de fosfatasa alcalina (EC 3.1.3.1). En el plasma de mujeres embarazadas, durante el último trimestre de gestación puede encontrarse otra isoenzima, la fosfatasa alcalina placentaria. Además, en extractos butanólicos de tejido placentario se ha encontrado una isoenzima unida a membrana, la fosfatasa alcalina placentaria de alto peso molecular. En suero de mujeres embarazadas se ha determinado la actividad de fosfatasa alcalina placentaria soluble pero, hasta el momento, no se ha detectado la presencia de la isoenzima de alto peso molecular. En nuestro laboratorio hemos desarrollado un método que permite la detección de fosfatasa alcalina de alto peso molecular en el pellet de plasma centrifugado a 100.000xg. Utilizando el mencionado método hemos determinado la actividad de fosfatasa alcalina placentaria de alto peso molecular en plasma de mujeres embarazadas durante el último trimestre de gestación

Valores normales de fosfatasa alcalina leucocitaria: estudio comparativo entre los métodos citoquímicos: Gomori-Takamatzu y Burstone / Normal value of leukocyte alkaline phosphatase: comparative study between citochemical methods Gomori-Takamatzu and Burstone

En 100 donantes de sangre sanos, de ambos sexos, con edades comprendidas entre 20 y 40 años fueron determinados los valores de fosfatasa alcalina leucocitaria (FAL). Se utilizaron los métodos citoquímicos: Gomori Takamatzu y el de Burstone. Con el primero se encontró un valor escor de 30 a 140, con un promedio de 75.68 y una desviación normal de 30.62. Con el segundo se encontró un valor escor mínimo de 22 y un máximo de 150; promedio de 83,15 y desviación normal 31.49. Ambos métodos son comparables y los resultados obtenidos son válidos como valores normales de nuestra población sana